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In most cases of DS, the extra chromosome is present as a result of the failure of normal chromosome segregation during meiosis. The nondisjunction event is maternal in 95% of cases, occurring primarily during meiosis 1 in the maturing ovocyte before conception. Despite the high prevalence of DS, the mechanisms underlying meiotic nondisjunction are not yet understood. Abnormal folate metabolism and mutation of the MTHFR (EC 1.5.1.20) gene have recently been described as a possible cause of DS (1). The aim of this study was to check the results obtained by James et al.(1) by studying a cohort of women carrying DS fetuses.

MATERIALS AND METHODS

For the control group, 107 volunteers were recruited (37 men, 70 women; median age 40 y, mean age 43.2 ± 17.7 y, range 17–90 y). All were shown to be free from clinical symptoms of vascular disease.

Amniocentesis was performed between 16 and 24 wk in pregnant women aged >35 y or if echographic signs of DS or abnormal serum hormonal markers were suggestive of this diagnosis. Selection criteria were induced abortion of a fetus with karyotypically confirmed full trisomy 21 without translocation. No DS mothers with live births were studied. The 85 DS mothers were aged 34.9 ± 5.0 y (range 20–47 y). DNA polymorphisms of chromosome 21 were used to determine the parental origin of the extra chromosome 21 and the stage at which the meiotic error occurred (2). Genomic DNA was extracted from lymphocytes from DS mothers by standard procedures. For genotype analysis, the MTHFR gene was amplified by PCR after restriction enzyme digestion with HinfI using the primers and conditions described by Goyette et al.(3). Total plasma homocysteine was determined by immunoassay (Abbott Laboratories, North Chicago, IL, U.S.A.) using serum obtained from DS mothers and 39 pregnant women with normal fetuses to check serum hormonal markers of trisomy 21. In all cases, informed consent was obtained from the pregnant women and volunteers of control group for biochemical or molecular biologic investigations. This study was approved by Institutional Review Board of Hôpital Necker.

RESULTS

The frequencies of the C/C, C/T, and T/T genotypes among the DS mothers with maternal nondisjunction did not differ from those of the control subjects (Table 1). If DS mothers were assigned to two groups according to age (> or <40 y) no difference was observed (χ2 = 0.35) between the two groups in MTHFR genotypes (Table 1). The stage of meiotic error was also used to separate DS mothers in two groups. Again, no difference (χ2 = 2.58) was observed between the two groups (Table 1). Total plasma homocysteine concentrations in the pregnant women are shown in Table 2. No differences (t test) were observed between the various groups of subjects (homozygous, heterozygous, or normal subjects for the 677C/T mutation) either in the control or DS mother groups.

Table 1 MTHFR polymorphism in DS and controls
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Table 2 Total fasting plasma homocysteine in DS mothers and control subjects
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DISCUSSION

MTHFR acts at a critical metabolic juncture in the regulation of cellular methylation reactions, catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the remethylation of homocysteine to give methionine. A frequent mutation, described as the thermolabile mutation (677 C/T MTHFR mutation), may modify enzymatic activity. The specific activity of MTHFR is low in the heterozygous C/T genotype and in the homozygous T/T genotype (35% and 70% of the C/C genotype, respectively). James et al.(1) described a significantly higher frequency of this mutation in 57 mothers of children with DS than in 50 control women. They recently extended their study (4) to 157 DS mothers and 140 control women and obtained similar results. Our results differ from those recently described (4). Some differences were observed between the groups of DS mothers studied. We took into account all DS cases, whereas James et al.(1, 4) studied only mothers of liveborn DS children. The percentage of the various genotypes in the controls was similar in the two studies. It is well known that the effect of the T/T genotype on plasma homocysteine concentration is related to folate intake (5), the T/T genotype only inducing hyperhomocystinemia if associated with a low intake of folate. Therefore, we determined total fasting serum homocysteine in DS mothers according to genotype. No hyperhomocystinemia was detected in the three groups of DS mothers. James et al.(1) found hyperhomocystinemia in the T/T DS mothers, but the homocysteine determinations were performed many months after the delivery and it is not clear whether hyperhomocystinemia occurred during pregnancy. Our determinations of total plasma homocysteine concentration were performed closer to conception (between 16 and 24 wk of pregnancy). Some differences between traditional French and American diets may account for the lack of hyperhomocystinemia in our subjects, even in the T/T genotype subjects. The search for the etiology of chromosome 21 nondisjunction is of great importance. It is therefore essential to discriminate clearly between the possible etiologic factors. Hyperhomocystinemia may be one of these factors, and it is probably of value to determine homocysteine in the plasma of pregnant women early during the pregnancy, along with hormonal markers, and to correlate plasma homocysteine concentration with the delivery of DS fetuses.